CN116064484B - Mutant chitinase SsChi18A-2 and application thereof - Google Patents

Mutant chitinase SsChi18A-2 and application thereof Download PDF

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CN116064484B
CN116064484B CN202211148739.8A CN202211148739A CN116064484B CN 116064484 B CN116064484 B CN 116064484B CN 202211148739 A CN202211148739 A CN 202211148739A CN 116064484 B CN116064484 B CN 116064484B
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mutant
chitinase
gene
chitin
sschi18a
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CN116064484A (en
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冀福全
王禄山
夏光明
吴秀芸
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Shandong Meimei Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/62—Plastics recycling; Rubber recycling

Abstract

The invention discloses a mutant gene SsChi18A-2 of chitinase SsChi18A, wherein the mutation site of the gene is E287A, the nucleotide sequence of the gene is shown as SEQ ID No.1, and the protein sequence is shown as SEQ ID No. 2. The invention also discloses application of the gene in degrading colloidal chitin and fungus dregs. Experiments prove that the activity of mutant chitinase coded by the gene is improved by 35% compared with wild type enzyme, and the activity of the mutant chitinase is improved by 49% compared with wild type enzyme when degrading fungal cell walls, and the results show that the mutant has wide application in degrading chitin substrates.

Description

Mutant chitinase SsChi18A-2 and application thereof
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to mutant chitinase SsChi18A-2 and application thereof.
Background
Chitin is poly-beta-1, 4-N-acetylglucosamine (NAG), which is the second most abundant polysaccharide in nature except cellulose. Chitin is mainly derived from crustaceans, and is produced in water body of up to 10 per year 11 Tons. The processes of demineralizing chitin and deproteinizing biomass to obtain pure chitin are usually performed with concentrated acids or bases, but can cause corrosion and environmental problems. Enzymatic degradation of chitin can produce a variety of pharmacological activitiesSex ingredients such as N-acetylglucosamine (NAG) and Chitooligosaccharide (CHOS) can act as drug delivery vehicles and antioxidants, playing a role in antitumor, wound healing, blood cholesterol control and food preservation. The chitinase has wide sources, the enzymolysis process is environment-friendly, and the application potential in industrial production is huge. At present, chitinase preparations are widely applied to various fields of medicines, foods, biotechnology, plant disease and pest control and the like.
Chitin is the most abundant natural polysaccharide in nature next to cellulose, and is polymerized from beta-N-1, 4-glucose and beta-N-1, 4-glucosamine, respectively. These polysaccharides are highly crystalline, and form a degradation-resistant barrier through complex hydrogen bonding networks both intramolecular and intermolecular, making degradation difficult. But organisms evolve complex mechanisms for efficiently utilizing crystalline polysaccharide, and the intractable polysaccharide is hydrolyzed into soluble polysaccharide by utilizing the synergistic effect of environment-oriented glycoside hydrolase, in particular to some soluble oligosaccharides with prebiotic value, which has important application value in the fields of industry, agriculture and medical treatment.
Streptomyces sp.F-3 is a thermophilic actinomycete isolated and screened by the inventor group at a high Wen Shengjing, and the optimal growth temperature of the thermophilic actinomycete is 50 ℃. The whole genome sequencing analysis result shows that the strain contains 9 chitin degradation related enzymes and contains one exo-persistent chitinase SsChi18A. The rich chitin degrading enzyme and thermophilic characteristic make it have the potential to develop into chitin degrading industrial bacteria.
In practical application of SsChi18A, the problems to be solved still exist; shortening the process flow and improving the production benefit has been highly demanded to have higher activity chitinase. To expand the industrial application of SsChi18A, increasing its enzymatic activity is a critical task.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides mutant chitinase and application thereof.
In the present invention, the mutated chitinase is named SsChi18A-2, and can also be named SsChi18A-E287A or E287A.
In one aspect, the invention provides a mutant chitinase having an amino acid sequence as set forth in SEQ ID No. 2.
In another aspect, the invention also provides a gene encoding the mutant chitinase; preferably, the sequence of the gene is shown as SEQ ID No. 1.
In another aspect, the present invention also provides a recombinant vector comprising the mutated chitinase encoding gene; preferably, the recombinant vector is a recombinant expression vector; preferred are vectors of the pET series, e.g.pET-15 b, pET-22b, pET-28a.
In another aspect, the present invention also provides a recombinant strain comprising the recombinant vector described above, preferably, the recombinant strain is escherichia coli, such as escherichia coli BL21.
In another aspect, the invention also provides the use of the mutant chitinase, the coding gene, the recombinant vector or the recombinant strain in degrading chitin-containing material. It is understood that chitin-containing materials include naturally chitin-containing materials, or processed chitin-containing materials; in addition, chitinase monomers are also a concept of chitin-containing materials.
In one embodiment, the chitin-containing material is a residue from a fungus, e.g., a residue of Aureobasidium pullulans.
In one embodiment, the Aureobasidium pullulans residue is the residue remaining after the fermentation of Aureobasidium pullulans to produce a specific product.
In another aspect, the invention also provides application of the mutant chitinase, the coding gene, the recombinant vector or the recombinant strain in preparation of chitooligosaccharides.
In another aspect, the invention also provides a method of degrading a chitin-or chitin-oligosaccharide-containing material, the method comprising the step of treating the chitin-or chitin-oligosaccharide-containing material with the mutated chitinase, the encoding gene, the recombinant vector or the recombinant strain. It is understood that chitin-containing materials include naturally chitin-containing materials, or processed chitin-containing materials; in addition, chitinase monomers are also a concept of chitin-containing materials. The chitooligosaccharide-containing material includes a natural chitooligosaccharide-containing material, or a processed chitooligosaccharide-containing material; furthermore, simple chitooligosaccharides are also a concept of chitooligosaccharide-containing materials.
Preferably, the oligosaccharide is a disaccharide, trisaccharide, tetrasaccharide, pentasaccharide or hexasaccharide.
Further, the temperature of the treatment is 30 ℃ to 80 ℃, preferably 50 ℃ to 70 ℃; the pH of the treatment is 3-11, preferably 4-7.
The invention has the beneficial effects that:
the activity of the mutant SsChi18A-2 of the chitinase SsChi18A disclosed by the invention is improved by 35% compared with that of a wild type enzyme, and the enzyme activity is improved by 49% compared with that of the wild type enzyme when the fungal cell wall is degraded, so that the results show that the mutant has wide application in the aspect of degrading chitin substrates. When used as industrial enzymes for medicine, food, biotechnology, plant disease and pest control and the like, can maintain higher activity in a wider environment, and specific applications include but are not limited to:
in the process of medicine production, the mutant chitinase can be used as a microbial inhibitor to prepare ophthalmic preparations, and can also be used as an additive of antifungal medicines to treat fungal diseases. In the food industry, the mutant chitinase can be used for producing chitooligosaccharides, and can be used as an antimicrobial agent and an immunopotentiator to activate host defense systems; but also can be used as therapeutic agent and food additive for osteoarthritis and rheumatic arthritis to regulate human health. In the biotechnology industry, this mutant chitinase may be used in conjunction with other cell wall degrading enzymes for the preparation of fungal protoplasts. In the process of preventing and controlling plant diseases, the mutant chitinase can be used as a biocontrol agent for preventing plant pathogens and pests, and can rapidly hydrolyze cell walls of plant disease fungi, so that cells are cracked, and pathogenic fungi are killed.
Drawings
FIG. 1 is a SDS-PAGE diagram of protein purification.
FIG. 2 shows the relative enzymatic activities of wild-type WT and mutant E287A using colloidal chitin as substrate.
FIG. 3 is a product spectrum of mutant E287A and wild-type WT degraded colloidal chitin.
FIG. 4 shows the relative enzyme activities of mutant E287A and wild-type WT degraded Aureobasidium pullulans residue.
FIG. 5 is a spectrum of the mutant E287A and wild-type WT degraded Aureobasidium pullulans residue product.
Detailed Description
The present invention is further described in conjunction with the drawings and detailed embodiments, which are set forth below in order to provide a preferred embodiment of the present invention, but not to limit the invention in any way, and any person skilled in the art may make modifications to the equivalent embodiments using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
EXAMPLE 1 mutation of chitinase SsChi18A and construction of recombinant vector
The chitinase SsChi18A gene is obtained from the NCBI database, and the SsChi18A gene is connected with the plasmid pET28A to obtain the pET28A-SsChi18A plasmid. Site-directed mutagenesis is carried out on key amino acid sites predicted by bioinformatics, mutant primers are designed by taking the recombinant plasmid as a template, and site-directed mutagenesis is carried out to obtain mutant chitinase SsChi18A-2, wherein the primer sequences are as follows:
E287A-sense：CCTACGCAGCGGGCGTCGAGGACTA
E287A-antisense：AGTCCTCGACGCCCGCTGCGTAGGT。
the nucleic acid sequence of the mutated chitinase SsChi18A-2 is shown as SEQ ID No.1, the amino acid sequence is shown as SEQ ID No.2, and the E at 287 th site is mutated into A relative to the wild type chitinase.
The nucleotide sequence of the finally obtained mutant plasmid pET28A-SsChi18A-E287A is shown in SEQ ID No. 3.
EXAMPLE 2 construction of recombinant engineering bacterium containing the mutant Gene SsChi18A-2
Constructing recombinant engineering bacteria containing the mutant gene SsChi18A-2, which comprises the following specific steps: adding 50 mu L of competent cells of escherichia coli BL21 into the mutant plasmid pET28A-SsChi18A-E287A with correct sequencing, and carrying out ice bath for 30min; heat-shock at 42 ℃ for 90s; ice-bath for 2min, adding 1mL of liquid LB, and culturing in a shaking table at 37 ℃ for 1-1.5h; centrifugation at 8000rpm for 2min, the supernatant was discarded (leaving a small amount of bottom liquid). The remaining solution was spread on LB plates containing 50. Mu.g/mL kanamycin, spread evenly until dry, and incubated overnight at 37℃with inversion; the next day, single clone is selected and inoculated in 5mL LB culture medium containing antibiotics, and cultured at 37 ℃ and 200rpm overnight, thus obtaining recombinant engineering bacteria containing mutant gene SsChi 18A-2.
EXAMPLE 3 recombinant expression of the mutant Gene SsChi18A-2
Taking the recombinant engineering bacteria containing the mutant gene SsChi18A-2, and fermenting and culturing in LB culture medium (containing 50 mug/mL kanamycin) at 37 ℃ and 200rpm until the OD600 = 0.6-0.8; adding IPTG with the final concentration of 0.2mM, and carrying out induction culture for 20 hours at 20 ℃; centrifugation is carried out at a speed of 8000rpm and a temperature of 4 ℃ for 10min to obtain bacterial precipitation, and cells are broken by ultrasonic after re-suspension; centrifuging to obtain supernatant (crude enzyme solution), and filtering the supernatant with a 0.22 μm filter head; the column packing was combined with the filtrate and prepared for affinity purification. The target protein was affinity purified using GE Healthcare HisCap Co 6FF resin (Smart-life Sciences, changzhou, china). Purified protein plus NaH at pH 5.0 2 PO 4 -citric acid buffer, ultrafiltration at 4900rpm at 4 ℃, to a filtered buffer ph=5.0; the concentration of the purified protein was measured using coomassie brilliant blue staining method.
As shown in FIG. 1, SDS-PAGE patterns after protein purification are shown, marker is 25.0, 35.0, 45.0, 66.2, 116.0kDa, and lanes are Marker, crude enzyme solution, precipitate, effluent, 10mM imidazole eluent, 20mM imidazole eluent, 100mM imidazole eluent, respectively. As can be seen from the figure, the band of the target protein is located around 45kDa, which corresponds to the protein size.
Example 4, enzymatic properties of mutant chitinase SsChi18A-E287A (SsChi 18A-2) compared to wild-type enzyme SsChi18A.
(1) Colloidal chitinase Activity assay
Diluting mutant chitinase SsChi18A-E287A and wild-type enzyme WT to 0.01mg/mL; 100. Mu.L of NaH at pH 5.0 was added to the control tube 2 PO 4 100. Mu.L of diluted enzyme solution was added to each tube of the experimental group, and 100. Mu.L of colloidal chitin (dissolved in 50mM NaH at pH 5.0) at a concentration of 20mg/mL was added to each tube 2 PO 4 -citric acid buffer), at 60 ℃ for 10min; 300 mu L of DNS is added into each tube, and the boiling water bath is carried out for 10min; rapidly cooling, shaking, centrifuging, collecting supernatant, and measuring OD540; three replicates were run for each enzyme; calculating the reducing sugar amount according to the standard curve, and then calculating the specific enzyme activity according to the formula; the enzymatic activities of the two proteins were compared.
The relative enzyme activities of the mutant chitinase E287A and the wild type chitinase WT under the optimal conditions are shown in the figure 2, and the enzyme activity of the mutant xylanase E287A is 1.35 times that of the wild type xylanase WT, which proves that the mutant chitinase has stronger enzyme activity on chitosans and more excellent application value.
(2) HPLC analysis of colloidal chitin degradation products
The mono-and disaccharide concentrations were determined using a High Performance Liquid Chromatography (HPLC) system using a Bio-Rad Aminex HPX-42C, 300X 7.8mm (catalog # 125-0096) and SPD detector (Kyoto Shimadzu, japan). Purified enzyme (0.01 mg/mL; 250. Mu.L) was reacted with colloidal chitin (20 mg/mL, 500. Mu.L) at 60℃for 30min. After completion, the solution was centrifuged to remove insoluble substrate, and 400. Mu.L was mixed with 100. Mu.L of acetonitrile to terminate the reaction. The obtained sample was stored at-20℃until high performance liquid chromatography. The mobile phase is 70% acetonitrile nano-purified water solution, and the flow rate is 0.2mL/min. The isolated product was detected with an SPD detector at 210 nm. All samples were in triplicate.
The product spectra of mutant chitinase E287A and wild type chitinase WT degrading colloidal chitin are shown in FIG. 3. The products of both were mono-saccharides (peak starting at 7 min) and disaccharides (peak starting at 9.5 min), with disaccharides being the main. In comparison, the SsChi18A-E287A products have relatively high levels of mono-and disaccharides.
(3) Enzyme activity determination of degradation fungus dreg
Diluting mutant E287A and wild-type enzyme WT to 0.1mg/mL; adding 1mL of diluted enzyme solution into the experimental group tube, adding 0.05mg of Aureobasidium pullulans residue, reacting at 60 ℃ for 1h, sampling 200 mu L, adding 300 mu L of DNS, and carrying out boiling water bath for 10min; rapidly cooling, shaking, centrifuging, collecting supernatant, and measuring OD540; three replicates were run for each enzyme; calculating the reducing sugar amount according to the standard curve, and then calculating the specific enzyme activity according to the formula; the enzymatic activities of the two proteins were compared.
The relative enzyme activities of mutant E287A and wild-type WT degrading Aureobasidium pullulans residue are shown in FIG. 4. The enzyme activity of the mutant E287A is 1.49 times of that of the wild type WT, which proves that the mutant chitinase has stronger enzyme activity on Aureobasidium pullulans residues and more excellent application value.
(4) FACE analysis of bacterial residue degradation product spectrum
Diluting mutant E287A and wild-type enzyme WT to 0.1mg/mL; adding 1mL of diluted enzyme solution into the experimental group pipe, adding 0.05mg of Aureobasidium pullulans residue, and reacting at 60 ℃ for 1h for sampling; detecting the product spectrum by adopting a FACE electrophoresis method: firstly, mixing supersaturated 7-amino-1, 3-naphthalene disulfonate (ANDS) solution dissolved in 15% acetic acid with the same volume of a sample (5 mu L of each solution), and carrying out light-shielding reaction for 1 hour; an equal volume of NaCNBH is then added 3 Adding the solution to the mixture and incubating overnight at 42 ℃; 15. Mu.L of a 50% sucrose solution was added to the mixture; and the labeled product was used for electrophoresis at a ratio of 7. Mu.L/well; the image is scanned using the chemidoc MP imaging system and the image file is stored in TIFF format.
The spectrum of the Aureobasidium pullulans residue product of mutant E287A and wild-type WT treated at 60℃for 1h is shown in FIG. 5.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.

Claims (12)

1. A mutant chitinase having an amino acid sequence as set forth in SEQ ID No. 2.
2. A gene encoding the mutated chitinase of claim 1.
3. The gene according to claim 2, wherein the sequence of the gene is shown in SEQ ID No. 1.
4. A recombinant vector comprising the gene of claim 2 or 3.
5. The recombinant vector according to claim 4, wherein the recombinant vector is a recombinant expression vector.
6. The recombinant vector according to claim 5, wherein the backbone vector of the recombinant vector is derived from pET-15b, pET-22b or pET-28a.
7. A recombinant strain comprising the recombinant vector of any one of claims 4-6.
8. The recombinant strain of claim 7, wherein the recombinant strain is escherichia coli.
9. The recombinant strain according to claim 8, wherein the recombinant strain is escherichia coli BL21.
10. Use of a mutant chitinase of claim 1, a gene of claim 2 or 3, a recombinant vector of any of claims 4-6 or a recombinant strain of any of claims 7-9 for degrading chitin-containing material.
11. Use of a mutant chitinase of claim 1, a gene of claim 2 or 3, a recombinant vector of any of claims 4-6 or a recombinant strain of any of claims 7-9 for the preparation of chitooligosaccharides.
12. A method of degrading a chitin-or chitin-oligosaccharide-containing material, the method comprising the step of treating the chitin-or chitin-oligosaccharide-containing material with the mutant chitinase of claim 1 or the recombinant strain of any one of claims 7-9.
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